Semiquantitative Epstein-Barr Virus (EBV) Polymerase Chain Reaction for the Determination of Patients at Risk for EBV-Induced Lymphoproliferative Disease After Stem Cell Transplantation

Abstract

Epstein-Barr virus–induced lymphoproliferative disease (EBV-LPD) is a serious and potentially fatal complication after allogeneic stem cell transplantation (SCT). To evaluate levels of EBV DNA in SCT patients, a semiquantitative polymerase chain reaction (PCR) assay was developed. DNA was extracted from peripheral blood leukocytes and diluted, and PCR was performed by using a primer set specific for a well-conserved sequence of the internal repeat 1 region of the EBV genome. Forty-one SCT patients were screened with this method. Thirty-seven patients received allogeneic transplants, of which 18 were T-cell–depleted marrow. Four additional patients received autologous SCT, one of which was T-cell depleted. The mean time of follow-up by EBV PCR was 147 days (range, 47 to 328 days) posttransplant. The range of EBV copies/μg DNA from normal EBV sero-positive donors was 40 to 4,000. Seven patients had ≥40,000 copies of EBV DNA/μg DNA, all of whom were recipients of T-cell–depleted SCT. Five of the seven patients with elevated levels of EBV DNA developed EBV-LPD. Four of these five patients with EBV-LPD had elevated levels of EBV DNA from 1 to 8 weeks before diagnosis. Two patients with EBV-LPD had normal levels of EBV DNA, and two patients with ≥40,000 copies EBV/μg DNA did not develop EBV-LPD. In one patient, clinical resolution of disease correlated with a decrease in EBV DNA and an increase in the level of EBV-specific cytotoxic T-cell precursors. These data indicate that the measurement of EBV viral load with semiquantitative PCR is useful in detecting EBV-LPD in high-risk patients before the onset of clinical symptoms. Because not all patients with elevated levels of EBV DNA develop EBV-LPD, semiquantitative PCR results cannot substitute for clinical, radiographic, and pathological confirmation of this diagnosis.

Use of EBV PCR for the Diagnosis and Monitoring of Post-Transplant Lymphoproliferative Disorder in Adult Solid Organ Transplant Patients

“…Early detection of EBV DNA in peripheral blood leukocytes or plasma using quantitative polymerase chain reaction (PCR) may provide an indirect method of identifying patients at risk for PTLD and to monitor their response to therapy. PCR assays for various EBV targets such as EBNA-EBV PCR for Diagnosis and Monitoring of PTLD 1, EBER-1, LMP have been used to identify patients at risk for PTLD, especially in the pediatric and allogeneic bone marrow transplant populations (11)(12)(13)(14)(15)(16). These two patient populations are particularly at high risk for PTLD, given the lack of EBV immunity in most pediatric patients and the high level of immunosuppression and the altered immune system seen in T-cell depleted or mismatched allogeneic bone marrow transplants.…”

Increased Ig‐null B lymphocytes in the peripheral blood of pediatric solid organ transplant recipients with elevated Epstein‐Barr viral loads

“…A high load of EBV in the peripheral blood (>1000 copies/10 5 PBMCs) is strongly associated with a diagnosis of PTLD (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). This load is at least 2-3 orders of magnitude greater than the viral load measured in normal individuals, and is detected 2-6 wk before patients present with symptoms of PTLD (16,20).…”

Immunotherapy for Epstein-Barr Virus-Associated Cancers in Children

“…PCR-based assays that measure viral load in PB or serum are powerful aids to monitor EBV reactivation. EBV viral load in PBMCs of patients post-HSCT is strongly correlated to the histopathological diagnosis and, more importantly, often precedes the clinical diagnosis [69][70][71]. For example, in a group of 26 pediatric T-cell-depleted allogeneic stem cell recipients, at least 2 weeks prior to clinical diagnosis a rapid increase in EBV viral load indicated developing PTLD (sensitivity 100%, specificity 95%) [72].…”

“…For example, in a group of 26 pediatric T-cell-depleted allogeneic stem cell recipients, at least 2 weeks prior to clinical diagnosis a rapid increase in EBV viral load indicated developing PTLD (sensitivity 100%, specificity 95%) [72]. However, some patients have normal EBV levels prior to diagnosis, while others with elevated levels do not develop PTLD [70]. In addition, the specificity of this type of monitoring varies between patient groups (nonmanipulated versus T-cell-depleted graft [73]) and applied PCR techniques.…”